DESCRIPTION: (Applicant's Abstract) Neurotransmitter levels in brain are critical for normal brain function. Abnormal levels of neurotransmitter, resulting in inappropriate neural signaling, underlie a diverse list of brain disorders. For example, epilepsy, excitotoxic cell death, depression, and a number of conditions related to drug abuse are all related to abnormal transmitter levels in brain. Neurotransmitter transporters are proteins, located on neurons and glia, that function in part to transport transmitter from the extracellular milieu into cells. As such, they play a central role in regulating synaptic signaling. While much is being learned about the permeation properties of various neurotransmitter transporters (e.g., transport rates, substrate affinities, conducting states), little is known about how particular structural domains of the transporter participate in the permeation process. Furthermore, while it is known that transporter function can be regulated, the extent to which the regulation occurs through manipulation of the domains involved in permeation is not known. The major goal of this application is to define the role of intra-molecular and inter-molecular interactions in regulation of the GABA transporter GAT1. Specific Aim 1 is to test the hypothesis that the amino terminal cytoplasmic tail of the GABA transporter GAT1 positively regulates GABA transport through interactions with internal cytoplasmic loops of the transporter. Specific Aim 2 is to test the hypothesis that proteins that physically interact with the amino terminal tail of GAT1 negatively regulate GAT1 function by preventing the N-terminal tail from interacting with the internal cytoplasmic loops of the transporter. Specific Aim 3 is to test the hypothesis that intermolecular interactions that negatively regulate transporter function can be modulated by physiologically relevant factors. These studies are important because they will (i) define a new regulatory role in permeation for intracellular transporter domains; (ii) elucidate a novel mechanism for the cellular regulation of transporter function through protein-protein interactions; and (iii) provide data that could be useful in strategies aimed at regulating transporter function in the treatment of disorders related to abnormal transmitter levels.